U.S. patent number 7,118,626 [Application Number 10/651,499] was granted by the patent office on 2006-10-10 for crystallization cassette for the growth and analysis of macromolecular crystals and an associated method.
This patent grant is currently assigned to University of Alabama in Huntsville. Invention is credited to Juan-Manuel Garcia-Ruiz, Jose A. Gavira-Gallardo, Greg Jenkins, Joseph D. Ng, Mark Wells.
United States Patent |
7,118,626 |
Ng , et al. |
October 10, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Crystallization cassette for the growth and analysis of
macromolecular crystals and an associated method
Abstract
The invention is a crystallization cassette and associated
method for growing and analyzing macromolecular crystals in situ by
X-ray crystallography. The cassette allows proteins (as well as
other macromolecules) to be crystallized by the counter-diffusion
method in a restricted geometry. Using this procedure, crystals can
be adequately prepared for direct X-ray data analysis such that the
protein's three-dimesional structure can be solved without crystal
manipulation.
Inventors: |
Ng; Joseph D. (Huntsville,
AL), Garcia-Ruiz; Juan-Manuel (Granada, ES),
Gavira-Gallardo; Jose A. (Granada, ES), Wells;
Mark (Athens, AL), Jenkins; Greg (Madison, AL) |
Assignee: |
University of Alabama in
Huntsville (Huntsville, AL)
|
Family
ID: |
34217414 |
Appl.
No.: |
10/651,499 |
Filed: |
August 29, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050045094 A1 |
Mar 3, 2005 |
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Current U.S.
Class: |
117/68;
422/245.1; 117/70; 117/69 |
Current CPC
Class: |
C07K
1/306 (20130101); C30B 29/58 (20130101); Y10T
117/1024 (20150115) |
Current International
Class: |
C30B
7/02 (20060101) |
Field of
Search: |
;422/245.1
;117/68,69,70 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Gavira et al., Ab Initio Crystallographic Structure Determination
Of Insulin From Protein To Electron Density Without Crystal
Handling, Acta Cryst., Jul. 2002, 58, pp. 1147-1154. cited by other
.
Ng et al., Protein Crystallization By Capillary Counterdiffusion
For Applied Crystallographic Structure Determination, Journal Of
Structural Biology, 142, pp. 218-231, 2003. cited by other.
|
Primary Examiner: Kunemund; Robert
Attorney, Agent or Firm: Alston & Bird LLP
Government Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
The United States Government may have rights in the inventions set
forth herein as provided by the terms of Grant No. NCC8-243 awarded
by the National Aeronautics and Space Administration.
Claims
What is claimed is:
1. A crystallization cassette comprising: a support member having a
top, middle, and bottom portion; a housing member joined to the top
portion; a stabilizing member joined to the middle portion having a
plurality of capillary passageways; a precipitating reservoir
member joined to the bottom portion, whereby the capillary tubes
are in fluid communication with the precipitating reservoir; and a
plurality of capillary tubes each having a proximal and distal end,
wherein the capillary tubes' proximal ends are each joined to the
housing member, and wherein each capillary tube extends downwardly
from the housing member through a capillary passageway.
2. A crystallization cassette according to claim 1, wherein the
precipitating reservoir member includes: a surface facing the
distal ends of the capillary tubes; and a plurality of cavities on
the surface, wherein each cavity respectively corresponds to a
capillary tube.
3. A crystallization cassette according to claim 1, wherein the
capillary tubes are suitable for X-ray diffraction.
4. A crystallization cassette according to claim 1, wherein the
capillary tubes are selected from the group consisting of quartz,
acrylic poly(methly methacrylate), polystyrene, mylar
polycarbonate, CR 39, copolymers of styrene and poly(methly
methacrylate), and their derivatives, and combinations thereof.
5. A crystallization cassette according to claim 4, wherein the
capillary tubes are quartz.
6. A crystallization cassette according to claim 1, wherein the
capillary tubes have a diameter from about 0.05 to 1 mm.
7. A crystallization cassette according to claim 1, wherein the
capillary tubes have a diameter about 0.3 mm or less.
8. A crystallization cassette according to claim 1, wherein the
support member is a shaft.
9. A crystallization cassette according to claim 2, wherein the
precipitating reservoir member further includes a pierceable layer
on the surface.
10. The crystallization cassette according to claim 9, wherein the
pierceable layer is selected from the group consisting of latex,
wax, plastic plug, agarose, and fracture ease.
11. The crystallization cassette according to claim 2, wherein the
plurality of cavities each have a volume from about 5 to 50
.mu.L.
12. A crystallization cassette comprising: a shaft having a top,
middle, and bottom portion; an upper member joined to the top
portion of the shaft; a middle member proximate to the middle
portion of the shaft including: a first channel for receipt of the
shaft; and a plurality of capillary passageways that are
substantially parallel to the shaft and extend longitudinally
through the middle member; a plurality of capillary tubes each
having a proximal and distal end, wherein the capillary tubes
extend downwardly from the upper member, and wherein each capillary
tube extends downwardly through a capillary passageways; and a
lower member proximate to the lower portion of the shaft having a
first surface having a plurality of cavities located on the
surface, wherein each cavity is in alignment and corresponds to a
capillary tube, a second surface, opposite the first surface, and a
second channel for receipt of the shaft, whereby the capillary
tubes are in fluid communication with the lower member.
13. The crystallization cassette according to claim 12, wherein the
shaft has a vertical axis extending from the lower member to the
upper member and a keyway extending longitudinally along the
axis.
14. The crystallization cassette according to claim 13, wherein the
lower member has a key disposed in the second channel such that the
key is adapted to slide along the keyway.
15. The crystallization cassette according to claim 13, wherein the
middle member has a key disposed in the first channel such that the
key is adapted to slide along the keyway.
16. The crystallization cassette according to claim 12, wherein the
upper member includes a plurality of independently pivotable
capillary housing sections, wherein each pivotable housing section
is joined to the proximal end of a capillary tube and has a hinge
disposed proximate to the shaft, and wherein the pivotable housing
section is adapted to pivot upwardly such that the capillary tube
is extended outwardly.
17. The crystallization cassette according to claim 16, wherein the
outwardly extended capillary is substantially perpendicular to the
vertical axis of the shaft.
18. The crystallization cassette according to claim 12, wherein the
middle member has an outer edge that is parallel to the vertical
axis, the outer edge having a plurality of channels that extend
laterally through the middle member from the outer edge to the
plurality of passageways such that a capillary tube is reversibly
insertable through the channel into the passageway.
19. The crystallization cassette according to claim 12, wherein the
capillary tube has a ferrel disposed proximate to the distal
end.
20. The ferrel according to claim 19, having a substantially
circular body with a lower and upper portion, and a base rim
disposed at the lower portion having a diameter larger than the
body.
21. The ferrel according to claim 20, having a beveled edge
disposed at the upper portion.
22. The crystallization cassette according to claim 19, wherein the
diameter of the passageways are the same or larger than the
diameter of the body and smaller than the diameter of the rim such
that as the middle disc is slid downwardly along the shaft the
bodies of the ferrels will slide into the passageways.
23. The crystallization cassette according to claim 12, wherein the
first channel is disposed at the center of the middle member.
24. The crystallization cassette according to claim 12, having a
positioning lock for holding the middle member stationary relative
to the shaft including: a positioning channel extending laterally
through the middle member from the outer edge to the first channel;
a positioning recess on the shaft that is perpendicular to the
vertical axis and is aligned with the positioning channel; and a
positioning pin that is reversibly insertable into the positioning
channel such that as the middle member is slidably moved along the
shaft the positioning pin can be inserted through the positioning
channel into the positioning recess to thereby lock the middle
member's position.
25. The crystallization cassette according to claim 12, wherein the
lower member has a pierceable layer disposed on the first
surface.
26. The crystallization cassette according to claim 25, wherein the
pierceable layer is selected from the group consisting of latex,
wax, plastic plugs, agarose, and fracture ease.
27. The crystallization cassette according to claim 12, wherein the
plurality of cavities each have a volume from about 5 to 50
.mu.L.
28. The crystallization cassette according to claim 12, wherein the
lower member has a first depth control member for controlling the
depth to which the capillary tubes are inserted into the
cavities.
29. The crystallization cassette according to claim 28, wherein the
first depth control member is a stud extending outwardly from the
first surface in a direction that is substantially parallel to the
vertical axis of the shaft.
30. The crystallization cassette according to claim 28, wherein the
lower member's orientation to the shaft is inverted so that the
second surface is facing the middle member.
31. The crystallization cassette according to claim 28, wherein the
lower member has a second depth controlled member on the second
surface for controlling the distance between the distal ends of the
capillaries and the second surface.
32. The crystallization cassette according to claim 31, wherein the
depth control member is a second stud having a height that is
greater than the height of the first depth control member, and
wherein the second stud extends outwardly from the second surface
in a direction that is substantially parallel to the vertical axis
of the shaft.
33. The crystallization cassette according to claim 12, wherein the
second channel is disposed at the center of the lower member.
34. The crystallization cassette according to claim 12, wherein the
capillary tubes are suitable for X-ray diffraction.
35. The crystallization cassette according to claim 12, wherein the
capillary tubes are selected from the group consisting of quartz,
acrylic poly(methly methacrylate), polystyrene, mylar
polycarbonate, CR 39, copolymers of styrene and poly(methly
methacrylate), and their derivatives and combinations thereof.
36. The crystallization cassette according to claim 12, wherein the
capillary tubes are quartz.
37. The crystallization cassette according to claim 12, wherein the
capillary tubes diameters are from about 0.05 mm to 1 mm.
38. The crystallization cassette according to claim 12, wherein the
capillary tubes diameters are about 0.3 mm or less.
39. The crystallization cassette according to claim 12, wherein the
upper, middle, and lower members are disc shaped.
40. The crystallization cassette according to claim 12, wherein the
cassette has 12 capillary tubes.
41. The crystallization cassette according to claim 12, wherein the
overall shape of the cassette is cylindrical.
42. The crystallization cassette according to claim 12, wherein the
shaft, upper member, and lower member are comprised of an amorphous
non-refractive plastic.
43. The crystallization cassette according to claim 12, wherein the
cassette is made from a material selected from the group consisting
of quartz, acrylic poly(methly methacrylate), polystyrene, mylar
polycarbonate, CR 39, copolymers of styrene and poly(methly
methacrylate), and their derivatives and combinations thereof.
44. A crystallization cassette according to claim 12, wherein the
plurality of cavities each respectively have a lower portion and an
upper portion.
45. A crystallization cassette according to claim 44, having a
pre-loaded lower member comprising: a capillary sealant disposed in
each cavities' lower portion; a precipitating solution,
cryoprotectant solution, and a high X-ray scattering atom component
disposed in the cavities upper portion; and a pierceable member
disposed on the first surface such that the cavities are
sealed.
46. A crystallization cassette according to claim 45, wherein the
cavity sealant is selected from the group consisting of wax and
clay.
47. A crystallization cassette according to claim 45, wherein the
precipitating solution is selected from the group consisting of
salts and alcohols.
48. A crystallization cassette according to claim 45, wherein the
cryoprotectant solution is selected from the group consisting of
primary alcohols, glycerol, polyethylene glycol, methylpentanediol,
and derivatives thereof.
49. A crystallization cassette comprising: a shaft having a top,
middle, and bottom portion; a capillary housing member joined to
the top portion of the shaft; a capillary stabilizing member joined
to the middle portion of the shaft having a first channel that is
adapted for slidably receiving the shaft, and a plurality of
passageways that are parallel to the vertical axis of the shaft and
extend longitudinally through the stabilizing member; a plurality
of capillary tubes having a proximal and distal end, wherein the
capillary tubes extend downwardly from the housing member, and
wherein each capillary tube extends downwardly through one of the
passageways; and a precipitation reservoir member joined to the
lower portion of the shaft having a surface facing the stabilizing
member, a plurality of cavities located on the surface, wherein
each cavity is in alignment and corresponds to a capillary tube,
and a second channel that is adapted for slidably receiving the
shaft.
50. A method of growing biological crystals comprising the steps
of: a. providing a crystallization cassette as described in claim
45 having a pro-loaded lower member; b. depositing a protein
solution on the pierceable layer above each cavity; c.
repositioning the lower member along the shaft until the distal
ends of the capillary tubes contact the protein solutions; d.
diffusing the protein solutions into the capillary tubes; e.
piercing the pierceable layer with the distal end of the capillary
tubes by repositioning the lower member towards the middle member;
f. contacting the distal ends of the capillary tubes with the
precipitating solutuion, cryoprotectant solution, and scattering
atom component; g. diffusing the precipitating solution,
cryoprotectant solution, and scattering atom component into the
capillary tubes; h. growing biological crystals in the capillary
tubes; i. sealing the distal ends of the capillary tubes with the
capillary sealant; and j. selecting crystals for X-ray
diffraction.
51. The method for growing biological crystals according to claim
50, wherein the step of providing a pre-loaded loaded member
further comprises the steps of: a. inserting a capillary sealant
within the cavities; b. inserting precipitating, cryoprotectant,
and scattering atom solutions into each of the cavities; and c.
sealing the cavities with a pierceable layer.
52. The method for growing biological crystals according to claim
50, further comprising the step of analyzing the crystals in
situ.
53. The method for growing biological crystals according to claim
52, wherein the step of analyzing crystals in situ further
comprises the steps of: a. mounting the cassette in an X-ray
diffractometer on a motorized adaptor for rotating the cassette; b.
extending a capillary tube outwardly from the cassette; c. applying
a stream of cryogenic gas to the capillary tube; d. applying an
X-ray beam to a desired location on the extended capillary tube; e.
rotating the capillary in the X-ray beam; and f. collecting x-ray
data.
54. The method for growing biological crystals according to claim
50, wherein the biological crystal is selected from the group
consisting of proteins, nucleic acids, and viruses.
55. The method for growing biological crystals according to claim
50, wherein the volume of protein solution and precipitating
solution are about a 1:1 ratio.
56. A cassette positioning system for rotating a crystallization
cassette about an x-axis and y-axis comprising: an X-ray source; a
turntable for rotating the cassette about the y-axis of rotation;
an eccentricity correction stage disposed below the turntable; a
capillary scan stage disposed under the eccentricity stage; and a
cassette rotation stage for rotating a the cassette about the
x-axis.
57. A method of growing and analyzing macromolecules comprising:
providing a plurality of capillary tubes each having a first and
second end; contacting the second end of each capillary tube to a
solution having a solvated macromolecule therein; diffusing the
solution into the capillary tubes; inserting the second end of each
capillary tube into a corresponding cavity having one or more
precipitating agents therein; allowing the one or more
precipitating agents to counter-diffuse against the solution in
each capillary tube; allowing macromolecule crystals to grow in the
capillary tubes; and analyzing the crystals in situ via x-ray
diffractometry.
58. The method according to claim 57, further comprising sealing
the second end of each capillary tube.
59. The method according to claim 57, wherein the macromolecule
comprises a protein.
60. The method according to claim 57, wherein the precipitating
agent further comprises a cryoprotectant solution, scattering atom
component, or combination thereof.
61. The method according to claim 57, further comprising disposing
a pierceable layer between the solution and each corresponding
cavity, and wherein the solution is in the form of a droplet.
62. The method according to claim 61, wherein the step of inserting
the second end of each capillary tube into a corresponding cavity
further comprises piercing the pierceable layer with the second end
of each capillary tube.
Description
BACKGROUND OF THE INVENTION
The recent deciphering of entire genomic sequences of different
organisms, including humans, has resulted in a demand to decipher
three-dimensional structures of protein gene products. Determining
the structures of proteins may allow researchers to compile
structural information that will facilitate predictions of function
for almost any protein from knowing its coding sequences. Gaining a
better understanding of protein structure and function may enable
drug researchers to develop new drug treatments that target
specific human, animal, and plant diseases. The human body alone
has an estimated 52,000 different proteins. Determining the
structures to atomic resolution for all these proteins is a
daunting challenge, at best. X-ray crystallography currently offers
one method to achieve this goal and is the only method to date for
determining macromolecules greater than 35,000 Daltons.
Today, advanced recombinant DNA methods, systematic approaches for
protein crystallization, and highly developed X-ray diffraction
instruments and procedures contribute to determining protein
structure. The limiting step in protein structural determination is
the ability to obtain protein crystals that are suitable for X-ray
diffraction. Suitable crystals should be able to diffract to atomic
resolutions greater than 3 Angstroms with reflections that can be
readily indexed.
The process for obtaining crystals suitable for X-ray diffraction
normally is divided into four discrete steps. The first step
includes determining conditions for initial protein
crystallization. There are numerous factors influencing crystal
formation, which include: pH, ionic strength, temperature, gravity,
and viscosity, to name but a few. Second, the initial
crystallization conditions are optimized to produce crystals that
are suitable for X-ray diffraction. This step entails making minute
adjustments to the many crystallization parameters to produce the
highest quality crystal.
In the third step, the crystals are treated with a cryoprotectant
solution so that the protein crystal will tolerate supercooled
conditions. Protein crystals are very sensitive to X-ray radiation
and therefore data collection must be performed under super cooled
conditions. During this step, the researcher typically tests the
crystal in a variety of cryogenic solutions at different
concentrations and soak times.
In the final step, strong X-ray scattering atoms are required for
ab initio phasing. Atoms such as sulfur or metal ions are intrinsic
to most proteins and are often used for crystallographic phasing by
using the atoms' anomalous signals. However, halides or heavy
metals provide much higher X-ray scattering signals for effective
phasing and they are typically incorporated into the protein in an
invasive manner. This step usually requires rigorous testing to
find appropriate scattering atoms that can isomorphously
incorporate into the crystal without damaging the crystalline
order.
Typically, the third and fourth steps require the researcher to
manually transfer protein crystals between different solutions
followed by mounting the crystal on cryoloops for X-ray analysis.
These steps require that the researcher delicately handle the
crystal because any over excursion of force or mishandling could
damage the crystal. Current methods for growing and analyzing
protein crystals are time intensive, often fail to produce useful
crystals, and require that the researcher use extreme care when
handling the crystals.
From the foregoing, it should be readily apparent that obtaining
protein crystals and their subsequent preparation for X-ray
analysis is a very time consuming and limiting step in determining
protein structure. Consequently, more efficient methods are needed
for growing protein crystals suitable for analysis.
BRIEF SUMMARY OF THE INVENTION
The invention is a crystallization cassette that is useful for
crystallizing biological molecules. Although the invention is
described for crystallizing proteins for X-ray analysis, it should
be understood that other macromolecules such as nucleic acids and
viruses are also be applicable. The cassette provides a technique
that incorporates all four protein crystallization steps and the
step of X-ray analysis into a single apparatus. All steps are
performed in situ so that it is not necessary for the researcher to
manually manipulate the crystals. The cassette is adaptable for
high-throughput crystallography so that the process can be
performed under automated conditions.
The cassette has a top section and a lower member. The top section
contains multiple capillary tubes that extend downward from a
housing member through passageways contained in a stabilizing
member. The tips of the capillaries extend below the stabilizing
member. The lower member contains multiple cavities that contain a
precipitating solution, cryoprotectant solution and a scattering
atom component. Each cavity corresponds to a single capillary.
A pierceable layer seals the desired solutions and scattering atom
component within the cavities. A drop of protein sample is placed
on the pierceable layer above each cavity. The top section is
attached to the lower member so that the capillary tips contact the
protein samples.
The protein samples are taken into the capillaries by capillary
action. After the protein solutions have filled the capillary tubes
to the desired levels, the lower member slides towards the
stabilizing member so that the capillary tips penetrate the
pierceable layer and the tips contact the solutions contained
within the cavities. At this time, the protein solutions and the
solutions contained within the cavities counter-diffuse against
each other and a supersaturation wave is formed within each
capillary. Protein crystals begin to form as the superstaturation
wave moves through the capillary.
During the counter-diffusion process, a spatial-temporal gradient
is formed along the length of the capillary tube. As a result,
varying supersaturation conditions that lead to crystal growth are
simultaneously present in the capillary. Each cassette will greatly
improve the chances of obtaining crystals that are suitable for
X-ray analysis. Thus, the invention is a significant step forward
in achieving the goal of solving the structures for thousands of
proteins.
In one embodiment, the housing member is designed to allow each
capillary tube to pivot upwardly so that a capillary's tip extends
outwardly away from the cassette. Each capillary tube can be
extended away from the cassette at an angle that varies anywhere
from about 0 to 180 degrees. The outward extended capillary is
placed in an X-ray beam for data collection. The capillaries are
analyzed while they are in the cassette or, alternatively, they can
be removed for individual analysis.
The capillary tubes are constructed of an amorphous material that
is suitable for X-ray analysis. This is usually a quartz or
amorphous polymer. The size of the capillary tube may vary
depending upon use and desired crystal size. The diameter can range
anywhere from about 0.05 mm to 1 mm, with capillary tubes having
diameters less than about 0.3 mm obtaining the best results.
The size and shape of the cassette can vary depending upon the size
of the capillaries or the number of capillaries that are used. A
cylinder shape normally offers the most efficient space packing
geometry.
Thus, the invention provides among other things, an apparatus for
growing, cryoprotecting, incorporating scattering atoms, and
analyzing macromolecular crystals in situ for direct protein
structure determination.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
Having thus described the invention in general terms, reference
will now be made to the accompanying drawings, which are not
necessarily drawn to scale, and wherein:
FIG. 1 is a perspective view of a crystallization cassette that has
been fully assembled;
FIG. 2 is a perspective view of the of the cassette shown in FIG. 1
having the lower member separated from the shaft;
FIG. 3a is a perspective side view of the cassette shown in FIG. 2
having the middle member disengaged from the ferrels;
FIG. 3b is a perspective view of the cassette shown in FIG. 2
illustrating that the middle member can slide along the shaft;
FIG. 3c is a perspective view of the cassette shown in FIG. 3b
having a capillary tube in an outwardly extended position;
FIG. 4a is an underside view of the upper member;
FIG. 4b is an underside view of the upper member having side
channels;
FIG. 5 is a perspective view of the cassette shown in FIG. 2 having
a positioning pin;
FIG. 6a is a perspective view of the lower member having a depth
control stop;
FIG. 6b is a perspective view of the lower member having an
alternative form of the depth control stop;
FIG. 7a is a perspective view of the cassette being assembled for
use and having protein drops deposited on the lower member;
FIG. 7b is a perspective view of the cassette shown in FIG. 7a that
has been assembled for use and is taking protein solution into the
capillary tubes;
FIG. 7c is a perspective view of the cassette shown in FIG. 7b
having the capillary tubes filled with protein solution;
FIG. 7d is a perspective view of the cassette shown in FIG. 7c
having the capillary tubes filled with protein solution and
illustrating the capillary tubes penetrating the pierceable layer
so that the tips are in contact with the precipitating
solutions;
FIG. 7e is a perspective view of the cassette shown in FIG. 7d
illustrating protein crystallization as the precipitation solution
diffuses across the capillary tubes;
FIG. 7f is a perspective view of the cassette shown in FIG. 7e
illustrating the step of sealing the capillary tubes by contacting
the capillary tips with the sealant;
FIG. 7g is a perspective view of the cassette shown in FIG. 7f
having sealed capillary tubes;
FIG. 8a is a perspective side view of a second form of the
crystallization cassette; and
FIG. 8b is a perspective view of the cassette shown in FIG. 8a
having the precipitating reservoir member separated from the
support member.
DETAILED DESCRIPTION OF THE INVENTION
The invention now will be described more fully hereinafter with
reference to the accompanying drawings, in which some, but not all
embodiments of the invention are shown. Indeed, the invention may
be embodied in many different forms and should not be construed as
limited to the embodiments set forth herein; rather, these
embodiments are provided so that this disclosure will satisfy
applicable legal requirements. Like numbers refer to like elements
throughout.
Referring more specifically to the drawings, for purposes of
illustration, but not of limitation, there is shown in FIG. 1 a
form of the crystallization cassette referred to generally as 10.
FIG. 1 illustrates a fully assembled cassette having a support
member 100, a housing member 200, a stabilizing member 300, a
precipitating reservoir member 400, and a plurality of capillary
tubes 500. With reference to FIG. 8, reference number 800 broadly
designates a second form of the cassette. FIG. 8 illustrates a
crystallization cassette having a support member 814, a housing
member 822, a stabilizing member 816, a precipitating reservoir
member 900, and a plurality of capillary tubes 850.
Referring back to the first form of the cassette 10, FIG. 1
illustrates a cassette having a support member that is a shaft 100
having a top portion 110, a middle portion 120 and a lower portion
130. The housing member 200, also termed the upper member, is
joined to the top portion 110 of the shaft. The stabilizing member
300, also termed the middle member is joined to the shaft proximate
the middle portion of the shaft 120. The precipitating reservoir
member 400, also termed the lower member, is located near the
shaft's lower portion 130.
As depicted in FIG. 1, the upper member 200 is joined to the
shaft's top portion 110. With reference to FIGS. 1 through 4, the
upper member 200 is depicted as having a plurality of capillary
tubes 500 extending downwardly from its lower surface 270 at
240.
The proximal ends of the capillary tubes are joined to the upper
member at 240. The tubes 500 extend downwardly from the upper
member through passageways 310 disposed in the middle member 300.
As illustrated in FIG. 2, the capillary tubes' distal ends 510
extend downwardly below the middle member and are positioned above
the cavities 410 on the lower member 400.
With reference to FIGS. 4a and 4b, the upper member's lower surface
270 is illustrated having a plurality of openings 240 through which
the proximal end of the capillary is inserted. After inserting a
capillary tube into an opening 240, a bonding agent, such as an
adhesive, can be inserted through the upper openings 210 on the
upper member's top surface or through the outer openings 230 that
are disposed on the outer edge (FIG. 2). The bonding agent secures
the capillary tubes within the upper member. However, it should be
recognized that it is not necessary to use a bonding agent and that
the capillary tubes could be frictionally joined to the upper
member or non-permanently stabilized by vacuum grease or modeling
clay.
FIG. 4b illustrates an alternative method of attaching the
capillary tubes to the upper member. As shown in FIG. 4b, each
capillary opening is connected to a channel 242. Manual pressure is
applied to the channel's sides to increase the size of the channel
until the distance is great enough to slip the proximal end of a
capillary tube through the channel and into the opening 240. Once
the capillary tube is positioned in the opening, manual pressure is
released and the capillary tube is fit tightly within the opening.
This method of installing the capillary tubes allows the tubes to
be easily removed and replaced.
As shown in FIG. 3c, each capillary tube may extend outwardly from
the crystallization cassette. The upper member 200 has a plurality
of capillary housing members 250 that are each independently
pivotable. As shown in FIGS. 1 through 4, the upper member has a
plurality of narrow separations 220 that separate the capillary
housing members from each other. These narrow separations allow the
capillary housing members to pivot independently from each other.
Near the center of the upper member, proximate to the shaft 100,
the upper member has an area of decreased thickness. This area of
decreased thickness functions as a hinge so that the capillary
housing members are able to pivot upwardly. As shown in FIGS. 4a
and 4b, the hinge 260 is the area represented between the dashed
lines.
As should be apparent, hinge thickness will change the angle degree
to which the capillary tube is free to pivot. The capillary tube is
possibly able to pivot anywhere from about 0 to 180 degrees, with
an angle of about 90 degrees being somewhat more typical. The hinge
thickness range is typically from about 0.010 to 1.0 mm.
Capillary tubes that extend outwardly from the cassette offer a
significant advantage. This allows the cassette to be analyzed in
an automated system, which will greatly increase the speed and
efficiency at which crystals are screened and analyzed. In
practice, the outwardly extended capillary is extended and
positioned into an X-ray beam.
The upper member can be joined to the shaft in a variety of
different ways. For example, by way of illustration and not
limitation, the upper member can be adhesively bonded to the shaft;
the shaft or upper member may have a taper that allows the upper
member and the shaft to be frictionally fit together; or a set
screw could secure the upper member. Alternatively, the shaft and
upper member could be fabricated using an injection molding process
so that they have a unitary body.
As shown in FIG. 1, the middle member 300 is disposed near the
shaft's middle portion 120. The middle member has a channel that
can receive the shaft so that the middle member can slide along the
shaft. With reference to FIG. 3a through FIG. 3c, the middle member
is shown positioned at various locations on the shaft. The middle
member has a plurality of passageways 310 that extend
longitudinally from the middle member's upper surface to its lower
surface. Each capillary tube 500 has a corresponding passageway
310. The passageways secure the capillaries in the middle member,
while at the same time allowing the capillaries' bodies to slide
freely through the middle member as the middle member is moved on
the shaft.
A ferrel 320 prevents the capillaries from having too much free
movement within the passageways. The ferrel 320 is disposed near
the distal end 510 (tip) of the capillary tube. As the middle
member is slid downwardly into a locked or engaged position, the
ferrels 320 enter the passageways 310 to securely hold the
capillary tubes. Typically, the ferrel has a body 320, a base 322,
and a beveled upper edge 326. The base typically has a diameter
greater than the diameter of the ferrel's body 320. This prevents
the base from entering the passageway 310, and thereby stops the
middle member's movement towards the lower member. As shown in FIG.
2, the ferrel's body 320 slips into the passageway 310 and the base
322 remains outside the passageway. The upper edge of the ferrel
has a bevel 326 to assist in seating the ferrel into the
passageway. Although not illustrated, it should be understood that
the middle member's lower surface at the passageways' edges could
also be beveled.
The figures and above text describe a ferrel having a substantially
round body. The primary function of the ferrels is to stabilize and
steady the capillaries within the middle member, and as such, it
should be recognized that a variety of different structures could
perform the same function, although not necessarily with equivalent
results. For instance, a square ferrel and passageway could
stabilize the capillary tubes. Alternatively, a piece of tape or
cushioning material, such as a foam, attached to the distal ends or
located within the passageways could provide stabilization.
The illustrations show that each passageway 310 has a corresponding
channel 330, which extends laterally from the outer edge to the
passageway. The channels, similar to the passageways, extend
longitudinally from the middle member's upper surface to its lower
surface. When the middle member is pushed upwardly along the shaft,
the ferrels are disengaged from the passageways (FIG. 3a). At this
position, the capillary tubes fit rather loosely within the
passageway. The channel 330 allows the broadside of a capillary
tube's body to pass from the passageway 310, through the channel
330, and into an outwardly extended position (FIG. 3c).
To secure the middle member to the shaft and prevent its movement,
the cassette may have a positioning lock. As shown in FIG. 5, a
positioning pin 340 or set screw is inserted into a positioning
channel 342 that extends laterally through the middle member in a
direction that is substantially perpendicular to the shaft. The
shaft has a corresponding recess into which the pin is insertable
at 344. The recess is located on the shaft at a point that
corresponds to the positioning channel's horizontal location and
places the middle member in the desired vertical orientation along
the shaft. Typically, the desired vertical orientation between the
middle member and the shaft is when the passageways are securely
fit over the ferrels (FIG. 2). Alternatively, the middle member
could be secured in a desired location by a taper that is formed on
the shaft or within the middle member's channel. It should be
understood that securing the middle member's location along the
shaft's axis is not limited to the above recited methods and a
variety of different techniques could be used.
As stated above, the lower member is disposed on the shaft
proximate to its lower portion. Similar to the middle member, the
lower member also has a channel for receiving the shaft. As shown
in FIGS. 3 and 4, the second channel 420 is disposed near the lower
member's center. The lower member has an upper surface 470 that
faces the middle member. A plurality of cavities 410 are disposed
on the surface 470. Each cavity 410 is aligned with, and,
corresponds to a capillary tube.
While in use, the cavities 410 act as reservoirs to contain the
precipitating solution, which can also include cryoprotecting
agents, heavy X-ray scattering atoms or any other additives. The
bottoms of the reservoirs are layered with a sealant that is inert
to the solutions and additives. The solutions are added
individually or are pre-mixed and then deposited in the cavity. The
cryoprotecting agents, heavy atoms and other additives can be
initially mixed together or added in sequence during the
crystallization process. As illustrated in FIG. 2, the cavities 410
have a lower portion 414 and an upper portion 412. Typically, a
capillary sealant material, such as wax or clay, is disposed in the
lower portion. The various illustrations depict a lower portion 414
that has a conical shape, but it should be understood that it is
not necessary for the lower portion to have any particular shape
and a flat surface would suffice.
The cavities upper portions 412 are filled with the various
solutions for crystallization, as described above. The size of the
cavities can be varied depending upon the size of the capillaries.
Typically, capillaries having diameters from about 0.05 mm to 0.3
mm would have reservoirs containing volumes from about 5 to 50
microliters respectively. However, it should be recognized that
other volumes could be useful, although not necessarily with
equivalent results. After the cavities are filled, a pierceable
layer 460 is placed on the surface 470 to seal the cavities.
In an alternative arrangement, the cavities are subdivided into two
or three layers. In this arrangement, a second pierceable layer
separates the cryoprotectant and precipitating solutions. The lower
layer contains the capillary sealant. The middle layer is deposited
above the sealant layer. The middle layer contains the
cryoprotectant solution and, if desired, the scattering atom
component. The upper layer contains the precipitating solution and,
if desired, the scattering atom component. The division between the
layers is created in several ways. One method is to physically add
the second pierceable layer to the cavities to create the
separation. Alternatively, the second pierceable layer is added
during the manufacturing or assembly process. Under this
arrangement, the lower member is made from an upper and lower
section. The lower section, containing the cavities' lower and
middle layers are filled with the sealant and a pre-selected
cryoprotectant and scattering atom component. Thereafter, the
second pierceable layer is placed on the lower section to seal the
cavities. Next, the upper section is joined to the lower section to
complete the assembly process. The assembly process can be
completed within the lab or at the assembly plant.
It is envisioned that the lower members 400 can be pre-loaded at an
assembly plant with various sealants, cryoprotectant solutions,
precipitating solutions, and scattering atom components. A
researcher may choose from a variety of pre-loaded lower members.
Having pre-loaded lower members may also greatly enhance the
efficiency and speed at which different protein crystallization
conditions are screened. Additionally, pre-loaded lower members are
ideally suited for high-throughput crystallization methods.
With reference to FIGS. 6a and 6b, a capillary depth stop control
is illustrated. The depth stop control functions to limit the lower
member's upward movement as it slides along the shaft. Limiting the
upward movement helps to ensure that the distal ends of the
capillaries are not damaged by contact with the lower member. FIG.
6a illustrates a depth stop control that has two pairs of posts
(studs) on opposite surfaces of the lower member. The upper studs
440a extend outwardly from the upper surface 470 at an angle that
is substantially perpendicular to the surface. The lower studs
450a, similar to the upper studs, extend outwardly from the lower
surface at an angle that is substantially perpendicular to the
lower surface. As shown in FIG. 6a, the upper stud 440a height is
different than the height of the lower stud 450a.
The depth control stop may come in a variety of different
arrangements and orientations. For instance, as illustrated in FIG.
6b, the depth stop controls may be a unitary structure, such as
rings 440b, 450b that project outwardly from the upper and lower
surfaces. Alternatively, a removable pin or series of pins
positioned on the shaft could function as a depth control stop. The
pin(s) could be removed after completion of each step, thereby
allowing the lower member to move along the shaft to the next
desired position.
In an automated or manually operated system it may be sufficient to
have a single depth control stop. In an automated system, the
system would reposition the lower member a controlled distance at
pre-determined intervals. The single depth control stop would
prevent the capillary tips from contacting the cavities' bottoms.
However, it should be recognized that having multiple depth control
stops could be desirable or necessary depending upon experimental
need.
In a cassette having two depth control stops on opposite surfaces,
the lower stud 450a and upper stud 440a work cooperatively. In the
first step, the lower member's lower surface is positioned on the
shaft facing the middle member. Drops of protein sample are
deposited on this lower surface in alignment with the capillary
tubes. The lower stud 450a allows the capillary tips to contact
protein samples deposited on the lower surface, but prevents the
tips from contacting the surface. After the protein samples have
filled the capillaries, the lower member is removed from the shaft
and then reinserted onto the shaft so that the lower member's upper
surface 470 is facing the middle member. Thereafter, the upper stud
440a prevents the tips from contacting the cavity through the
remaining crystallization steps.
As shown in FIGS. 1 through 6, the shaft has a keyway 140 that
extends along its vertical axis from the bottom portion towards the
top portion. The middle portion and the lower portion each have a
corresponding key 350, 430, respectively. The keys are adapted to
slide within the keyway so as to prevent the middle or lower
members from rotating around the shaft, which would place the
passageways and cavities out of alignment with their respective
capillary tubes. Alternatively, the shaft could have a key and the
middle and upper members a keyway. It should be recognized that
changing the geometric shape of the shaft and corresponding
channels, such as to a square, would also prevent rotation about
the shaft.
As illustrated in FIG. 1, the crystallization cassette 10 has a
general cylindrical shape with the upper, middle, and lower members
being discs. The cylindrical shape of the crystallization cassette
10 normally provides for the most efficient packing of capillary
tubes. It should be recognized that the cassette could have a
variety of different sizes and shapes. As illustrated in FIG. 3,
the cassette 10 has 12 capillary tubes evenly spaced around the
shaft. Changing the overall size of the cassette will allow the
user to vary the number of capillary tubes that are present on the
cassette. For instance, the larger the cassette, the more capillary
tubes that can be placed in the cassette, and the opposite is
equally true for a smaller cassette.
The cassette can be fabricated to be useful with a variety of
different capillary tubes. Typically, the capillary size can range
from about 0.05 mm to about 1 mm in diameter. Capillary tubes
having diameters about 0.3 mm or less usually produce the best
results. Larger capillaries can be used, but as the capillary
diameter increases, the convective forces present within the
capillary also increase. If it is desirable to use capillaries
larger than 0.3 mm, a gel can be used to minimize convective flow
within the capillary. Commonly used gels for macromolecular
crystallization include agarose, silica gels, and polyacrylamides.
The capillary tubes are usually made of quartz because of its
amorphous qualities. Other materials may be used provided that they
are amorphous or near amorphous and do not contribute to
experimental diffraction.
The top section of the cassette, including the shaft, upper member,
middle member, and capillary tubes can be fabricated as a single
unitary structure through the use of injection molding. Materials
for manufacturing the cassette and the capillaries include, without
limitation, quartz, acrylic poly(methly methacrylate), polystyrene,
mylar polycarbonate, CR 39, copolymers of styrene and poly(methly
methacrylate), and derivatives or combinations of the
aforementioned materials.
The lower portion 414 of the cavity is loaded with a capillary
sealant that is suitable for sealing the capillary tubes' distal
ends. Typically, the sealant is a soft wax or clay. However, other
sealants could be used to seal the capillaries, although not
necessarily with equivalent results.
After depositing the sealant in the cavities lower portion, the
precipitating solution, cryoprotectant solutions, and scattering
atom component are deposited in the cavities' upper portions 412.
The solutions are either premixed or added individually to the
cavities.
The precipitating agent most commonly contains salts (e.g. ammonium
sulfate, sodium chloride or sodium citrate at concentrations of
about 2 3M), alcohols (e.g. ethanol, proponal, methylpentanediol at
concentrations of about 35 75%), or different forms of polyethylene
glycol (PEG) (e.g. PEG 4000, 6000, 8000 concentrations between 15
50%) in a buffered media. The volume of precipitating solution
(including any additional additives) placed into the cavities can
be as little as the equivalent volume of the protein solution
contained within the capillary.
Most protein crystals are very sensitive to X-rays and will not
survive the X-ray exposure that is necessary for data collection.
As a result, the crystals should be super-cooled without allowing
the solvent content to go through an ice transition before X-ray
analysis. Typically, this is accomplished by subjecting the crystal
to a stream of cryogenic vapor (with temperatures around -150 to
-170 C.), such as that from liquid nitrogen. For the sake of
simplicity, the supercooled crystals will be also referred to as
frozen crystals. In order for the crystal to endure the cooling
process it is treated with a cryoprotectant prior to freezing. The
cryoprotectant solution should protect the crystal while still
sustaining its ability to diffract X-rays. Examples of
cryoprotectants include glycerol, multiple alcohols, polyethylene
glycols, oils, and even Indian cooking butter. Useful oils are
typically composed largely of glycerides of the fatty acids, such
as oleic, palmitic, stearic, and linolenic. Other chemical
substances can be used provided that they sufficiently protect the
crystal's structure during freezing, there is no resulting
interference with crystal nucleation, and the crystal's ability to
diffract X-rays is not adversely affected.
In order to grow protein crystals that are adequate for ab initio
phase determination it is necessary to find a strong scattering
atom that is intrinsic to the protein or to incorporate a
derivative scattering atom, such as a heavy metal or halide.
Bromide and iodide are halides that have been shown to be useful
for diffusing into protein crystals and have been successfully used
in crystallographic phasing. The anomalous X-ray scattering signals
of halides are strong enough to provide phase information for X-ray
crystallography, and as such, are usually useful for incorporation
into the protein.
After the cavities are filled with the sealant and solutions, a
pierceable layer 460 is placed over the cavities to seal them. The
pierceable layer functions as a membrane to seal the solutions in
the cavities while at the same time allowing the capillaries'
distal ends to penetrate and enter the cavity. Suitable materials
for the pierceable layer include, without limitation, latex,
plastic plugs, waxes, agarose, or fracture ease.
Next, a drop of protein solution is deposited on the pierceable
layer in a position that is directly above each cavity. The volume
of protein solution deposited above each cavity will vary depending
upon capillary size. It is expected that a capillary that is about
0.3 mm or less in diameter requires a protein drop that is from
about 10 to 50 .mu.L. Higher volumes of protein solution can be
used, but greater volumes may adversely affect the diffusion
gradient within the capillary. Optionally, agarose can be added to
the protein sample at a low concentration. Agarose decreases the
convective mass transport within the capillary, helps to avoid
slippage of the crystal during initial screening, and facilitates
nucleation.
With reference to FIGS. 7a through 7g, the loading and use of the
cassette is illustrated. FIG. 7a depicts a fully loaded cassette
being assembled. As shown in FIG. 7a, a sealant 620 is in the lower
portion of the cavity 410, the upper portion is filled with the
desired precipitating solutions 610, the cavities are sealed with
the pierceable layer 460, and a protein drop 600 has been deposited
on the pierceable layer above each cavity.
FIGS. 7b and 7c illustrates the protein solution entering and
filling the capillary tubes. Capillary action takes the protein
solutions into the capillaries. In FIG. 7d the top section of the
cassette is moving downwardly towards the lower member so that the
capillary tips 510 penetrate the pierceable layer 460 and contact
the precipitating solutions 620.
Although the figures illustrate the cassette in a vertical
orientation during the soaking process, crystallization is best
performed in the horizontal orientation. The cassette uses
counter-diffusion to create a supersaturation wave as the solutions
diffuse against each other. In a vertical orientation any crystals
that are formed would have a tendency to fall downward in the
capillary disrupting any concentration gradients as well as mixing
different crystals.
FIG. 7e illustrates that as the precipitating solution diffuses
across the protein solution a supersaturation wave is formed and
protein crystals 630 begin to form. When the precipitating solution
(salt solution) contacts the protein solution, a liquid-liquid
free-diffusion system is formed activating a super saturation wave
along the capillary. This gradient is a result of the salts
initially diffusing into the protein solution, forming a salt
gradient of high concentration near the protein-precipitating
interface and falling to a lower concentration as it moves across
the capillary. As a result of the gradient, crystallization
conditions are not uniform throughout the capillary and crystals of
varying quality and size will be produced. Thus, one advantage of
the cassette is that multiple crystallization conditions are
present in a single capillary tube.
With time, the protein and precipitating solutions equilibrate.
Single crystals are typically readily observable within 3 to 7 days
of equilibration. The crystals should be sufficiently cryoprotected
within 1 to 4 weeks of equilibration depending on the
cryoprotectant and protein solutions.
With reference to FIG. 7f, the step of sealing the capillaries is
illustrated. As FIG. 7f illustrates, the top section of the
cassette is pushed towards the lower member until the capillary
tips contact the sealant 620. In this regard, FIG. 7g illustrates
capillary tips 510 that have been sealed with the sealant 640.
After the capillaries are sealed, the top section is removed from
the lower member and transferred to an X-ray diffractometer for
initial screening and data collection.
The cassette's design is particularly suited for attaching it to a
cassette positioning system. In the cassette positioning system,
the cassette is attached to a rotating adaptor that positions each
capillary tube in front of an X-ray beam for diffraction analysis.
The cassette positioning system is capable of rotating the cassette
about both the x-axis and y-axis. At the rotation stage, the
cassette is mounted so that its shaft is aligned in the x-axis.
After mounting the cassette in the rotating adaptor, an automated
mechanism indexes to a particular capillary so that the capillary
is aligned parallel to the X-ray beam. Next, the automated system
slides the middle member towards the upper member until the ferrels
are disengaged from the middle member. A capillary tube is extended
outwardly from the cassette until it is parallel to the y-axis of
rotation and is placed in alignment with an X-ray beam source.
The cassette rotation stage is disposed beneath a Y-Z translation
stage called the cassette scan stage. The cassette scan stage
translates the cassette so that the X-ray beam moves up and down
the capillary. As a result, the cassette positioning system is
capable of moving up and down in a direction that is parallel to
the y-axis of rotation. This allows a particular crystal in the
outwardly extended capillary to be aligned with the X-ray beam.
The Y-Z translation stage (the capillary scan stage) is disposed
beneath an X-Z translation stage called the eccentricity correction
stage. This stage centers the crystal in the X-ray beam.
The final stage is called the capillary rotation stage. The
capillary rotation stage rotates the entire system, including the
outwardly extended capillary tube about the y-axis. The capillary
rotation stage is a turntable that is disposed above the other
stages and is joined to the eccentricity correction stage such that
the system and cassette are suspended downwardly from the turntable
or any orientation that best adapts to the particular X-ray source.
The assembly is mounted on the turntable such that the center of
the capillary is aligned with the turntable's center of
rotation.
The cassette positioning system uses computer-controlled
microsteppers to translate and rotate the capillary through the
X-ray beam. During initial screening experiments, the cassette scan
stage will translate the capillary through the X-ray beam, pausing
in steps. At each step the eccentricity correction stage translates
the capillary back and forth across the beam to screen for high
quality crystals. During this step, the scattered X-ray intensity
of reflections over the background noise (I/sigma) within a
specific range of resolution is measured (usually reflection spots
having I/sigma measurements greater than 3 are preferred). The
quality of each crystal will be the evaluation by their I/sigma,
mosaicity of reflection spots and diffraction limit (greater than 3
Angstroms). If no high quality crystals are found, the cassette
positioning system advances the cassette to the next capillary and
the process is repeated.
If a crystal having sufficient quality for X-ray analysis is
detected, the targeted crystal is positioned in the center of the
X-ray beam and the axis of rotation for the turntable runs through
the crystal's center. The capillary can then be subjected to
super-cooling by a direct cryogenic gas flow to freeze the crystal.
The targeted crystal can then undergo a series of diffraction at
incremented angular oscillations until a complete data set is
obtained. Usually a 90 degree sweep in steps of 1 degree is
sufficient to collect a complete data set for high symmetry
crystals. Once the analysis is complete, the capillary is returned
to its beginning position within the cassette and the cassette is
rotated about the x-axis until the next capillary is aligned with
the X-ray beam. At this time, the process is repeated until all
capillary tubes have been analyzed.
The cassette's features make it particularly suited for
high-throughput protein crystallization. Robotic automated systems
could be used to take the cassette through multiple operations
making it possible to perform tests on thousands of samples
simultaneously. Numerous lower members could be manufactured and
pre-loaded with varying precipitation solutions. This would allow a
researcher to select and purchase lower members that are ready for
installation onto the shaft without requiring the need to transfer
solutions to the cassette. The cassette is adaptable to a robotic
system that could deposit a predetermined amount of protein
solutions on the lower member's surface, load the lower member onto
the shaft, and take the cassette through the steps necessary for
protein crystallization by repositioning the lower member on the
shaft at predetermined time intervals. After completing the protein
crystallization steps, the automated system transfers the cassette
to an X-ray diffractometer for analysis and data collection.
The advantage of the crystallization cassette is that it
simultaneously combines the processes of protein crystallization,
high X-ray scattering atom incorporation, and cryoprotection. This
allows the crystals to remain in a stable environment at all times
and eliminates the need for physical manipulation or exposing the
crystals to drastic chemical changes. After crystal growth is
completed the crystals can be quickly evaluated in situ without
ever having to remove them from their original growth
environment.
With reference to FIGS. 8a and 8b, a second embodiment of the
crystallization cassette 800 is illustrated. The cassette has a
support member 814, a housing member 812 joined to the top portion
of the support member, a stabilizing member 816 joined to the
middle portion of the support member, and a precipitating reservoir
member 900 (reservoir) that is joined to the support member's
bottom portion, and a plurality of capillary tubes 850 that extend
downwardly from the housing member through passageways 824 disposed
in the stabilizing member.
The capillaries' distal ends (tips) 852 extend through the
passageways and downwardly below the stabilizing member. In this
regard, FIGS. 8a and 8b illustrate the capillary tips extending
below the stabilizing member. Optionally, the support member has a
pair of feet 818 that help stabilize the cassette 800.
The reservoir member 900 has a plurality of cavities 920 on the
surface 940 facing the capillary tubes. Each cavity is aligned with
and corresponds to a single capillary tube. The cavities, similar
to the cavities discussed above, are reservoirs for the sealant
layer and the precipitating solutions.
The methods for using the first 10 and second cassette 800 are
substantially the same. Both cassettes use the counter-diffusion
technique to combine the four protein crystallization steps. With
reference to the method for using the cassette 800, the bottom
portion 914 of the reservoir member is filled with a sealant, such
as clay or wax. Next, A precipitating solution is added to the
upper portion 912 of the cavity. Placing a pierceable layer 950
over the cavities seals the cavities. A drop of protein solution is
then deposited on the pierceable layer above each cavity.
After completing the initial loading, the reservoir member 900 is
slid onto the support member and slid upwardly until the capillary
tips contact the protein sample. Capillary action takes the protein
solution into the capillary tubes. After an effective amount of
time has passed for the protein solution to fill the capillaries,
the reservoir member is slid upwardly until the capillary tips
penetrate the pierceable layer and contact the precipitating
solutions. The precipitating solutions and protein solution
counter-diffuse against each other until equilibration is reached.
After crystal formation and a sufficient amount of time has passed
to cryoprotect the crystals and to incorporate scattering atoms
into the protein crystals the capillaries are removed for X-ray
analysis.
As should be evident from the foregoing discussion, the
crystallization cassette is a beneficial tool to a
crystallographer. The cassette's design facilitates testing
multiple precipitating solutions and crystallization conditions
simultaneously. Its compact size and restricted geometry make the
cassette adeptly suited for easy transport and high-throughput
crystallization processes. The cassette is adaptable to automated
processes from the initial crystallization steps to the analysis
procedures performed on an X-ray diffractometer. As such, the
cassette is a valuable tool that will aid crystallographers in
deciphering and solving the structures for thousands of
proteins.
Many modifications and other embodiments of the inventions set
forth herein will come to mind to one skilled in the art to which
these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
* * * * *